1. Field of the Invention
The invention relates to the delivery of electromagnetic energy to living tissues, particularly in conjunction with the infusion of pharmaceutical agents. Apparatus and methods are provided for applying electromagnetic energy, by establishing field strength and current conditions including certain variations in the electrical parameters, and for measuring field and current parameters locally. The invention also relates to electrical stimulation (“electrostimulation”) of host tissue to enhance in vivo cellular delivery of pharmaceuticals, such as nucleic acids and other pharmaceutical entities, including but not limited to proteins and small organic or inorganic molecules, and for assessing the effects.
An aspect of the invention relates to electrostimulation of host tissues, especially skeletal muscle, which relies on application of an electric stimulus utilizing partially insulating electrodes and similar arrangements that limit current amplitude in the tissue. The current in the electrostimulation site preferably is limited to less than the tissue would conduct, due to its electrical resistance, if placed in direct conductive contact with electrodes at a particular potential difference. This can be accomplished in alternative ways according to the disclosure.
The invention provides enhanced delivery and/or expression of a transgene of interest while also minimizing certain undesirable effects such as involuntary muscle movements associated with the use of conducting electrodes.
2. Description of the Related Art
Studies have shown that applied electrical energy can affect a biological membrane, in that a sufficient application of energy increases the permeability of the membrane and thus allows solutions to diffuse through a membrane or tissue more readily to achieve a desired effect. Generally, this phenomena is associated with iontophoresis, electrophoresis or electroporation (collectively “electrical stimulation” or “electrostimulation”).
Iontophoresis generally concerns the introduction of an ionized substances through an intact membrane such as the skin, by application of a direct electric current. The current presumably entrains the ions and/or increases ion mobility in the tissue. Electrophoresis concerns the migration of ions in a fluid or gel under influence of an electric field. In electroporation, an electric field (often pulsed) and the associated induced current, induce microscopic pores to form in a membrane, typically a cell membrane. These pores are commonly called “electropores” and the process of forming them is electroporation. A potential application of electroporation is that solutions such as pharmaceutical agents, molecules, ions, and/or water can pass more readily from one side of the membrane to the other through the electrically generated pores. The pores preferably persist temporarily during application of the field. After application of the field, the pores should close or heal within a short period of time. However, the healing time is dependant on the amplitude and duration of the electrical stimulation, and it is possible to damage tissue permanently by application of too high an instantaneous power level and/or too long a duration of stimulation. The damage could be due to formation of untenably large or numerous pores, or resistive heating of the tissue, or both.
Electrically induced pores are readily observed in vitro. Cells in a solution are substantially independent of one another and are exposed to view. However, it may be difficult to observe temporary electropores in an in vivo setting, assuming that they occur. More and less-conductive tissues surround any given cell and often have an orientation peculiar to the tissue type. Thus discontinuities in conductivity presumably affect the manner in which electromagnetic energy is coupled to tissues, locally affecting the voltage gradient and current density. Tissues surrounding an observation site in vivo also would interfere with visual observation. Perhaps for these reasons, no exemplary in vivo study of electroporation is currently known to the inventor.
Without a relatively detailed understanding of the pertinent operative parameters, it may be difficult to assess and potentially to apply electrical stimulation (“electrostimulation”) of tissues to useful ends. Assuming that current and voltage are the primary operational parameters of interest, there are still innumerable ways in which current and voltage might be applied. A particular voltage or current might prove desirable or a particular power level might be needed. The voltage, current and/or power may have minimum and maximum values or a particular relationship. A time varying component might be critical, and various waveforms might be tried, at a frequency from DC (direct current) into radio frequencies. A time varying electrical stimulation might also prove beneficial for one purpose or another, for example varying a pulse rate, duty cycle, AC frequency or the like. The frequency, pulse rate, duty cycle or the like might be linearly varied, periodic or exponential. Periodic wave forms may or may not have a DC bias, and can be shaped as sine waves, sawtooth or triangle waves, square waves, square pulses of any desired duty cycle, exponentially-decaying or charging pulses, etc. Any of these waveform types might be applied continuously or in bursts or pulse trains. It would be advantageous to determine the effects of these different possibilities and to identify particular combinations that have a potentially useful application.
In electrical stimulation of tissues, contact and non-contact apparatus are possible. In a contact apparatus, a signal is applied by physically contacting a target tissue site using conductive electrodes attached on opposite sides of the target site. In a non-contact apparatus, an electric or magnetic field can be generated using electrodes or coils that are likewise disposed on opposite sides of the site. In the contact example, the tissue may have a reactive component (capacitance or inductance) and the conductivity of the tissue may change over time due to the effects of the application of energy (e.g., due to heating), but in general the electrical response of the tissue is according to Ohm's law. The current conducted through the tissue is proportional to the voltage, the specific proportion being the resistance of the tissue. There are inherent limitations in this fact. Assuming conductive contact, one normally cannot independently control the applied voltage without a corresponding effect on current, and vice versa. Increasing voltage and/or current in tissues lead to increased joule heating and potential spasmodic muscular contraction. In a non-contact example (limited to an externally applied electric field), little current is conducted, although there may be an increase in ion mobility and oscillation, depending on frequency.
Although electroporation, iontophoresis, electrophoresis and the like have been identified, there is little real understanding of the parameters involved. Attempts to make use of the phenomena have had mixed results. There has been little indication of a clear direction for development. It would be advantageous to improve understanding of these phenomena and to make progress in the development of protocols for administering pharmaceutical agents to tissues under electromagnetic influence. It would be most advantageous if the electrical and biological aspects were understood to the extent that protocols could be suggested for treatments involving specific pharmaceutical agents. To date, attempts to optimize electrical stimulation to achieve a desired result have been limited to empirical adjustments, for example of pulse parameters. Empirical adjustments can be an unsure proposition. Such empirical adjustments may logically assume that electrical energy at higher power levels achieves more extensive pore formation and thus better results than at lower levels. However, this is not a direct relationship and in any event there are drawbacks to increasing output power, such as potential gene integration, tissue damage and discomfort for the patient or host.
An improved method is needed for controlling, measuring and assessing the performance of pharmaceutical agent delivery systems utilizing in vivo electrical stimulation, that can address the needs to apply an optimal signal while preserving the host's comfort and avoiding integration and tissue damage. Such a system needs the capability to vary the application of energy in a manner that is variable over a useful range of voltage, current, waveshape, duty cycle, cadence or repetition and other factors. The system also should accurately measure the voltage and current levels under load from the tissues so as to monitor and potentially to control the application of electrical stimulation at the appropriate micro or macro level. The system should advantageously produce sampling information or otherwise communicate meaningfully with processes that permit correlation of the electrical parameters to the effectiveness of the treatment. The effectiveness of the treatment in that context should be assessed beyond the time of treatment, by means other than monitoring electrical parameters per se. Preferably, the system should be optimized for planning and testing electrical parameters, including the testing of options which are sensitive to considerations that are ancillary to the formation of pores in membranes. Such ancillary considerations may include, for example, the potential for gene integration, tissue damage or the comfort of the host (subject). The system and its testing facilities and methods should be optimized for adapting the technique to treatment using particular therapeutic agents, namely by facilitating the planning and testing of iontophoresis and electrophoresis regimes in connection with a variety of therapeutic agents.
WO 98/43702 (see also Mathiesen, 1999, Gene Therapy 6: 508-514) disclose in vivo electrostimulation of skeletal muscle within a calculated electric field strength ranging from about 25 V/cm to about 250 V/cm. The electric field strength was calculated simply as a two dimensional voltage gradient, namely the potential difference (V) between the conductive electrodes, divided by the distance (cm) between the electrodes. The discussion does not delve into the electrical current resulting at a given voltage, from conductive coupling of electrodes to the tissue, or how or why the voltage gradient and the current density might advantageously be distributed, or how these factors might affect charge migration or other considerations that could conceivably have an effect on the technique.
WO 99/01158, WO 99/01157 and WO 99/01175 disclose the use of low voltage for a long duration to promote in vivo electrostimulation of naked DNA. An electric field strength or voltage gradient of about 1 V/cm to about 600 V/cm is disclosed, depending upon the target tissue. This encompasses a relatively expansive range from minimal effect to potentially injurious levels. However, even higher voltage gradients have been proposed.
U.S. Pat. No. 5,810,762, U.S. Pat. No. 5,704,908, U.S. Pat. No. 5,702,359, U.S. Pat. No. 5,676,646, U.S. Pat. No. 5,545,130, U.S. Pat. No. 5,507,724, U.S. Pat. No. 5,501,662, U.S. Pat. No. 5,439,440 and U.S. Pat. No. 5,273,525 disclose electroporation/electrostimulation methodology and related apparatus wherein it is suggested that a useful electrical field strength range within the respective tissue is from about 200 V/cm to about 20 KV/cm. U.S. Pat. Nos. 5,968,006 and 5,869,326 further suggest that electric field strengths as low as 100 V/cm are useful for certain in vivo electrostimulation procedures.
Jaroszeski et al. (1999, Advanced Drug Delivery Reviews 35: 131-137) review the present landscape of in vivo electrically mediated gene delivery techniques. The authors emphasize previous success with delivery of chemotherapeutic agents to tumor cells and discuss some of the early results in this field.
Titomirov et al. (1991, Biochem Biophys Acta 1088: 131-134) delivered two plasmid DNA constructs subcutaneously followed by electrical stimulation of skin folds, generating an electric field strength from 400 V/cm to 600 V/cm.
Heller et al. (1996, FEBS Letters 389: 225-228) delivered plasmid DNA expressing two reporter genes to rat liver tissue by generation of high voltage pulses (11.5 KV/cm) rotated through a circular array of electrodes.
Nishi et al. (1996, Cancer Res. 56: 1050-1055) delivered plasmid DNA expressing a reporter gene to rat brain tissue. The authors utilized an electric field strength of approximately 600 V/cm.
Zhang et al. (1996, Biochem. Biophys. Res. Comm. 220: 633-636) delivered plasmid DNA transdermally to mouse skin with 120V pulses to the skin folds wherein the distance between the electrodes was only about 1 mm.
Muramatsu et al. (1997, Biochem. Biophys. Res. Comm. 223: 45-49) reported transfection of mouse testis cells with plasmid DNA via 100 V pulses with a 10 mS pulse duration.
Rols et al. (1998, Nature Biotechnology 16(2): 168-171) reported transfection of mouse tumor cells with plasmid DNA by applying voltages from about 300 to 400 V across a 4.2 mm spacing of the electrodes.
Aihara and Miyazaki (1998, Nature Biotechnology 16: 867-870) reported in vivo expression of (β-gal in mouse muscle tissue by delivering a square waveform pulse (50 mS duration) at constant voltage (60V) with the distance between the electrodes being 3-5 mm.
Vicat et al. (2000, Human Gene Therapy 11: 909-916) show that high voltage (900 V), short pulse (100 μS) electrostimulation protocols result in prolonged expression within targeted cells, in this case mouse muscle cells.
Widera et al (2000, J. Immunology 164: 4635-4640) apply 100 volts over a 5 mm distance with conducting electrodes to deliver hepatitis B surface antigen, HIV gag and env encoding DNA vaccines in vivo to mouse and guinea pigs.
Generally, the teachings of the prior art lack a rigorous investigation of the formation of electropores in tissue from the aspect of an electrical circuit, wherein the tissue is treated as a load to which a signal is applied. The application of electrical power to the tissue can be characterized not only by a coupling of electrical power to the tissue at a given voltage gradient, but also has other aspects. These include but are not limited to the current coupled to the tissue, which together with voltage determines power dissipation, how the coupling is effected spatially, which determines current distribution and in particular local current density, and various issues of timing. Furthermore, the prior art fails to adequately address ancillary aspects of the treatment, such as the muscle contractions that can be induced with the application of current to tissue. Such aspects can render a treatment tolerable or intolerable from a clinical perspective.
The foregoing prior art shows that relatively vigorous voltages and correspondingly substantial currents (based on the electrical resistance of the tissue) have at times been studied for potential effects on gene expression. Possible tissue damage concerns may favor using arrangements with modest electrical power dissipation in the tissue. However, despite work in the field of low voltage-based electrostimulation of skeletal muscle with conducting electrodes, there remains a need to eliminate the unpalatable features associated with the process, including severe involuntary muscle movements, while obtaining any biological advantages of the process. There also remains a need to distinguish and refine the operative parameters of the treatment, including by analysis of the process as an electrical circuit with the tissue coupled electrically to the signal source in particular ways and with a volume of tissue, and/or parallel conductive paths in the tissue, being treated as the electrical load. The present invention provides apparatus and methodology to address and meet these needs.